Analysis of the genetic variability of the 1st (CCC/ACC, P52T) and the 10th exons (bp 1012-1704) of the TSH receptor gene in Graves' disease.

We determined the genetic variability of the 1st (CCC/ACC, P52T polymorphic variant) and 10th exons (bp 1012-1704) of the TSH receptor (TSHR) gene in Graves' disease. A total of 101 Graves' patients and 163 control subjects were screened. The A253 mutant allele was carried by nine patients with Graves' disease (8.91%) and 13 control subjects (7.98%) in heterozygous genotype. No significant difference in the frequency of the mutant allele was found between Graves' patients and control subjects. These results provide evidence that the A253 polymorphism has no genetic relevance in Graves' disease. Moreover, the DNA nucleotide sequence of 693 bp of the 10th exon (bp 1012-1704) of the TSHR gene was determined in 15 Graves' patients. Six patients were homozygous for the wild-type allele and nine were heterozygous for the mutant allele at the 253rd nucleotide of the first exon. No polymorphism was found in the DNA sequences obtained from leukocytes of Graves' patients, similarly to the sequences obtained from the nine control subjects. None of the nine patients carrying the A253 polymorphism in the 1st exon of the TSHR had polymorphism in the examined part of the 10th exon, including two additional patients whose thyroid tissue was directly analysed. In all likelihood, the polymorphisms of the examined regions of either the 1st or the 10th exon of the THSR gene do not contribute to the genetic susceptibility to Graves' disease.

Effect of anti-thyroid peroxidase (TPO) antibodies on TPO activity measured by chemiluminescence assay.

A chemiluminescence method was developed to measure thyroid peroxidase (TPO) activity and the inhibitory effect of anti-TPO antibodies in purified porcine TPO. The TPO preparation was characterized kinetically and controlled by Western-blotting technique. The chemiluminescence method proved to be reproducible and much more sensitive than the widely used guaiacol method, being able to detect TPO concentrations of 2.21 x 10(-5) g/L vs 6.63 x 10(-2) g/L with the latter. Otherwise, the determinations with the two methods correlated well (r = 0.76). Investigating the effect of IgGs from 23 hypothyroid patients on measured TPO activity, we detected inhibition in 19 cases with the chemiluminescence technique (15 with the guaiacol method). Anti-TPO antibodies showed competitive inhibition of TPO activity with respect to the substrate guaiacol. In both systems, the inhibition is present in the IgG F(ab')2 fragment. We conclude that the high sensitivity of chemiluminescence detection allows routine determination of the inhibition of TPO activity by anti-TPO antibodies.

[Polymorphism of the 52 triplet gene (nucleotide 253) of the TSH receptor in Basedow-Graves patients and in healthy controls]. / A TSH-receptor gén 52. tripletjének (253. nukleotid) polimorfizmusa Basedow-Graves-kóros betegekben és egészséges egyénekben.

It has been analysed the polymorphism of the first exon of TSH receptor gene, at the first nucleotide of the triplet 52 of the genomic DNA (CCC/ACC, Pro/Thr) in Graves' disease and control population and was not found connection between the genetic background, clinical picture and some immunological parameters. Genomic DNA was obtained from formalin-fixed, paraffin embedded thyroid tissue of patients with Graves' disease underwent subtotal thyroidectomy and from peripheral blood leukocytes. The first exon and small part of the first intron of the TSH-R gene was amplified by PCR. The motif was amplified by the primers and primers includes restriction site of the Tth 111 I restriction enzyme and mutant form of the examined nucleotide. Genomic DNA having only wild allele was detected at 189 bp, while mutant allele was digested a 167 and a 22 bp fragments. Three persons (two female and one male) of the 32 patients with Graves' disease had mutation A253 in heterozygotic form: The patient with mutation A253 had no any symptoms of Graves' ophthalmopathy. No correlation was found between mutation A253 and the levels of anti-TSH-R-, anti TG, anti-TPO and anti-eye muscle antibodies. Surprisingly, it was found mutation of heterozygous genotype in two control individuals (one female and one male) of 14 without any symptoms of thyroid disease and negative laboratory findings. Finally, it has not been found the association of the 253 nucleotide of the codon 52 polymorphism with Graves' disease and ophthalmopathy and the allele A253 has no pathogenetic relevance in Graves' disease. On the other hand, it cannot be excluded that other mutation or sequence polymorphism in the remainder of TSH-R extracellular domain might be important in autoimmune mechanism of Graves' disease.

IgA autoantibodies against human eye muscle antigen detected by western blotting and immunohistochemical methods in Graves' disease.

The presence of IgA autoantibodies against human eye muscle was investigated in 40 patients with Graves' disease (33 had ophthalmopathy). IgA anti-eye muscle antibodies could be demonstrated in sera of patients using western blotting and immunohistochemical methods. For the detection of sera possessing autoantibodies against eye muscle antigen the indirect immunosorbent assay had been used. IgA anti-eye muscle antibodies could be demonstrated in 25 cases and IgG types in 16 cases out of 40 patients. These anti-muscle autoantibodies were associated with eye muscle rather than skeletal muscle, the number of positive cases with the latter being 5/40 for IgG and 2/40 for IgA. Immunoreactive bands of IgA autoantibodies against eye cytosol were found at 84, 64, 45, 40 and 25-23 kDa in 22, 2, 16, 2 and 18 cases, respectively. A difference was observed in the staining of IgG and IgA types of autoantibodies by immunohistochemical analysis of eye muscle tissue. The IgA anti-eye muscle antibodies reacted with muscle fibers and the IgG types showed staining on endomysium. No sera of Graves' disease patients gave staining on skeletal muscle tissue. The results supported the presence of IgA anti-human eye muscle antibodies in patients with Graves' ophthalmopathy, which might play a relevant role in the development of eye disease.

Kinetic characterization of thyroid peroxidase enzyme and its inhibition by autoantibodies.

A guaiacol method was developed to measure thyroid peroxidase (TPO) activity and the inhibitory effect of anti-TPO antibodies using purified porcine TPO. The TPO preparation was characterized kinetically and identified by western-blotting technique. The K(M) for guaiacol determined in vitro was 5.6 x 10(-4) M. Investigating the effect of IgG's from 23 hypothyroid patients on TPO activity inhibition was detected in 15 cases with the guaiacol method. It was found that anti-TPO antibodies exerted a competitive inhibition TPO activity with respect to the substrate guaiacol. The inhibition is carried by IgGF(ab')2 fragment. We wanted to gain a method which is interpreted immunologically and can be well employed in the clinical practice.